Dear Research Community,
I am encountering a significant hurdle in my research involving enzyme inhibition testing. The inhibitor I am investigating exhibits solubility exclusively in DMSO, rendering it insoluble in aqueous environments such as the 100mM phosphate buffer I am utilizing for enzyme kinetics studies. Upon attempting to incorporate the DMSO-dissolved inhibitor into the reaction mix, it precipitates out, leading to haze formation in the solution and hindering accurate data collection.
I am seeking insights and suggestions on how to effectively address this challenge. Specifically, I am interested in methodologies , or alternative solvents that could facilitate the integration of the inhibitor into the reaction mix without inducing precipitation. Additionally, any advice on modifying experimental conditions or buffer compositions to mitigate this issue would be greatly appreciated.
Thank you in advance for your expertise and assistance.
You may be able to improve the aqueous solubility of this substance by changing to a different buffer. 100 mM phosphate buffer has a high ionic strength, which is bad for aqueous solubility of hydrophobic compounds. Try a lower concentration (25 mM) of an organic buffer, such as Tris or HEPES, if the pH is in the 7-8 range. (Obviously, the choice of buffer depends on the pH.) You should re-check the optimization of your assay conditions when you change buffers.
Although it may be tempting to add a detergent to solubilize the substance, I would discourage this, because the concentration of detergent sufficient to keep it in solution will be higher than the critical micellar concentration, so that the compound will be sequestered in micelles, not free to bind to the enzyme.
Many enzymes can tolerate a few % of DMSO, and this much can slightly improve the aqueous solubility of hydrophobic compounds.
Ultimately, you will most likely have to modify the chemical structure of the inhibitor series to improve its solubility.
Dear Paul
As far as I know, the solubility of DMSO in water (e.g. phosphate buffer) at room temperature (20-25 °C) is around 65% (w/w). As you reported, the inhibitor molecule you have used is soluble in this solvent. But, you have encountered difficulty in realizing your inhibition assay. I guess that you have used the solvent at a relatively low concentration, thus to avoid denaturation of your enzyme. Is it true? To deal with you and try to find a solution, the inhibitor chemical formula must be indicated, unless you want to keep it unknown. Try to realize your inhibition assay at a high temperature (e.g. 40 °C), since the inhibitor molecule solubility increases in function of temperature. Also, you can experiment another solvent, as isopropanol. Besides, you can perform your assay under moderate agitation, thus to keep the assay mixture in a soluble-like state.
In addition, the suggestion of Adam Shapiro, to lower the phosphate buffer concentration (e.g. from 100 mM to 50 mM ) can be helpful. So, revise your assay conditions.
I have used firstly Dimethylformamide to improve aqueous solubility of peptides inhibitors/or substrates. After then, the dilutions of the peptide solutions are carried out with aqueous buffer - sodium acetate or phophate (0.05 M).
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